There is a continuous need in medical practice, research and diagnostic procedures for rapid, accurate and qualitative or quantitative determinations of biological substances which are present in biological fluids at low concentrations. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, prostaglandins or infectious organisms in blood, urine, saliva, vaginal secretions, dental plaque, gingival crevicular fluid and other biological specimens has to be determined in an accurate and rapid fashion for suitable diagnosis or treatment.
To provide such determinations, various methods have been devised for isolating and identifying biological substances employing specific binding reactions between the substance to be detected (identified herein as a "specific binding ligand") and a compound specifically reactive with that substance (sometimes identified as a "receptor" for the ligand).
The complex formed between ligand and receptor can be detected by a variety of known methods. The most commonly used methods employ a signal-generating moiety of some type which is either already attached to one of the components of the complex, or becomes part of the complex through further reaction. For example, in the formation of a complex of biotin with avidin, the complex may be detected using a label on either the avidin or biotin molecule. Such a label can be a radioisotope or an enzyme conjugated with the avidin or biotin. Alternatively, the avidin-biotin complex might be detected by further reaction with a labeled molecule which is specific to either or both parts of the complex. It is commonly known to do the same with antigens and their corresponding antibodies.
Preferred labels in specific binding reactions are clearly enzymes because the handling and disposal problems associated with radioisotopes can be avoided. Many enzymes are known to be useful in this context with peroxidases being the most common.
In diagnostic tests designed to be rapid and easy to use with moderate training in a doctor's office or clinic, the specific binding ligand of interest (such as an antigen from an infectious agent) is often detected using colorimetric or chemiluminescent signals resulting from reaction of the enzyme label with its corresponding substrate. There is a need to produce the signal quickly and intensely if the ligand is present.
However, there is also a need to have the signal produced in a defined region of a test zone in a test device so an adjacent or surrounding region could be used as a background control. In such cases, production of the signal should be modulated or stopped after a certain period of time in order to provide clear distinction between test zone and background zone.
An advance in the art in the detection of microorganisms associated with periodontal diseases is described and claimed in U.S. Ser. No. 468,392 (filed Jan. 22, 1990 by Snyder). This case describes the simultaneous detection and differentiation of these microorganisms, and particularly Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis (formerly known in the art as Bacteroides gingivalis) and Prevotella intermedia (formerly known as Bacteroides intermedius), in an assay using water-insoluble reagents in defined regions of a microporous filtration membrane. The simultaneous detection and differentiation of these microorganisms have considerable clinical and commercial significance.
The assay described in this copending application is carried out using peroxidase as a label on antibodies specific for the bacterial antigen. It is well known in such assays to use a buffered solution of sodium azide to stop the production of colorimetric signal when peroxidase reacts with a suitable substrate. In most instances, a low concentration (for example, 0.1 weight percent) of sodium azide performs satisfactorily.
However, it was determined that high pH (9 or more) washings were required to reduce background from nonspecific binding of target antigen with non-specific antibodies. Yet, when high pH wash solutions were used, sodium azide became ineffective to adequately modulate or stop the formation of signal, especially when the signal was detected on a porous microporous filtration membrane. In other words, the colorimetric signal production is only slowed down slightly by the azide and is not stopped. This causes the specific signal and the surrounding region on the membrane to continue to develop signal thereby reducing the case of interpretation and preventing accurate quantitation of positive results. It is desired to prevent this from happening, yet it is uncertain as to why the high pH wash affects the function of the sodium azide. However, it appears that the interaction of the membrane and the absorbent underneath it allows high pH wash solution to flow back into the region where signal is generated and the function of the azide is impaired.
It was considered to use greater concentrations of sodium azide and strong low pH buffers to solve the problem, but this was not acceptable because sodium azide is a well known toxic material and also has explosive properties.